AKTUALIZOVÁNO: duben 2006AKTUALIZOVÁNO: duben 2006 11

Expression of Estrogen Expression of Estrogen Receptor Receptor αα and and ββ in the Brain in the Brain

and Relationship with and Relationship with Behavior, Behavior, CCognitionognition, , MemoryMemory

and Stress in Health and and Stress in Health and Neurodegenerative diseaseNeurodegenerative disease

*Radka MOLIKOVA , *Marcela BEZDICKOVA,*Radka MOLIKOVA , *Marcela BEZDICKOVA, **Shan Shan HUANG, **He LI, **Shan Shan HUANG, **He LI, *****Zdenka *Zdenka

MICHALIKOVAMICHALIKOVA

* Dept. of General Anatomy, Faculty of Medicine, Palacky University Olomouc, Czech Rebublic * Dept. of General Anatomy, Faculty of Medicine, Palacky University Olomouc, Czech Rebublic EuropeEurope

** Division of Histology, Tongji Medical College, Huazhong University Science and Technology, ** Division of Histology, Tongji Medical College, Huazhong University Science and Technology, Wuhan P.R. of ChinaWuhan P.R. of China

***** * Medical Graphic Designer, Medical Graphic Designer, Faculty of Medicine, Palacky University Olomouc, Czech Rebublic Faculty of Medicine, Palacky University Olomouc, Czech Rebublic EuropeEurope

22

IntroductionIntroduction ERERαα and ERand ERββ are an important form of steroid are an important form of steroid

hormones with multiple targets, in the body and hormones with multiple targets, in the body and brain, and exerting ubiquitous effects on brain, and exerting ubiquitous effects on behavior. behavior.

In the brain these receptors are regionally In the brain these receptors are regionally specific, but both have widespread distributions, specific, but both have widespread distributions, which are largely non-overlappingwhich are largely non-overlapping[1[1, 7, 8, 7, 8]]

. .

In the CNS, estrogens are well known to cause a In the CNS, estrogens are well known to cause a importance for control of importance for control of REPRODUCTIONREPRODUCTION, , including including SEXUAL BEHAVIORSEXUAL BEHAVIOR but not only in but not only in the context of reproduction, but also with the context of reproduction, but also with reference to reference to COGNITION AND MEMORYCOGNITION AND MEMORY[1[1, 4, 6, 4, 6]]

..

The physiological function of ERThe physiological function of ERαα and ERand ERββ is under is under intense study but certain results indicate that ERintense study but certain results indicate that ERαα and ERand ERββ havehave different or event opposite biological different or event opposite biological actionsactions[1][1]

..

Hoon´s preliminary studies show that hormone Hoon´s preliminary studies show that hormone receptors, receptors, estrogen receptors (ERs),estrogen receptors (ERs), and cAMP and cAMP response elements binding protein (CREB) are response elements binding protein (CREB) are present in the mitochondrial matrix of neurons and present in the mitochondrial matrix of neurons and ER and CREB could be regulated by novel signaling ER and CREB could be regulated by novel signaling pathways in the intact central nervous system and pathways in the intact central nervous system and that their functions in the mitochondria might be that their functions in the mitochondria might be important in neuronal survivalimportant in neuronal survival[[1010]]

. .

PurposePurpose

On the basis of newly emerging complexities of On the basis of newly emerging complexities of estrogen's mechanisms of action it is importantestrogen's mechanisms of action it is important[1[1, 7, 7]]

: :

1.1. To distinguish To distinguish which pathwayswhich pathways of of ERERαα and ERand ERββ

are are involved in modifyinginvolved in modifying which behavior, which behavior,

cognition and memory.cognition and memory.

2.2. To To study the mechanisms by which estrogens study the mechanisms by which estrogens

mediatemediate behavior, cognition and memory in behavior, cognition and memory in

which parts of the brain during increasing which parts of the brain during increasing ((pEGFP-pEGFP-

C2- ERC2- ERαα,,ββ)) and decreasing and decreasing (siRNA) (siRNA) expression of expression of

ERERααand ERand ERββ. .

3.3. To To compare mediate mechanismcompare mediate mechanism and biological and biological

function of function of ERERααand ERand ERββ in the brain and in the brain and

potential relation to potential relation to neurodegenerative neurodegenerative

diseasesdiseases (PD, AD, HD). (PD, AD, HD).

ERERαα is localized on human chromosome 6, in is localized on human chromosome 6, in contrast to ERcontrast to ERββ, which sits on chromosome , which sits on chromosome 14.14. ERERαα and ERand ERββ thus represent two separate thus represent two separate gene products and share relationshipgene products and share relationship[1[1, 6, 6]]..

EERRαα is highly expressed in the classical is highly expressed in the classical estrogen target tissues such as uterus, estrogen target tissues such as uterus, mammary gland, bone and cardiovascular mammary gland, bone and cardiovascular systems, whereas ERsystems, whereas ERββ is mainly expressed in is mainly expressed in non-classical tissues such as prostate, ovary non-classical tissues such as prostate, ovary and urinary trackand urinary track[2][2]..

Overall distribution of EROverall distribution of ERαα and ERand ERββ in in different tissues figure 1different tissues figure 1[1][1]..

ERERαα and ERand ERββ

Figure 1

Gustafsson A.J. 1999 Estrogen receptor β-a new dimension in estrogen mechanism of action. Journal of Endocrinology 163, 380. [1]

ERERαα isis widely expressed widely expressed in the in the hypothalamushypothalamus. The distribution of ER . The distribution of ER ααconcentrated in the band of Broca, the concentrated in the band of Broca, the medial mamillary nmedial mamillary nuucleus, the medial cleus, the medial preoptic area, the paraventricular preoptic area, the paraventricular nucleus,nucleus,aand the ventromedial nucleusnd the ventromedial nucleus[3][3]..

ERERαα has been localized in the has been localized in the hippocampushippocampus, with dorsal vs. ventral , with dorsal vs. ventral differences in the ERdifferences in the ERαα expressionexpression[[55]]..

Sensitive binding study have also showed Sensitive binding study have also showed the the expressionexpression of estrogen receptors in of estrogen receptors in cells of the cells of the neocortexneocortex, where their , where their accurate localizationaccurate localization and and function is function is not so clearnot so clear (resp.unknow), but may (resp.unknow), but may involve mediation of estrogen action on involve mediation of estrogen action on sexual behaviorsexual behavior[4][4]. .

Neurodegenerative Neurodegenerative diseasedisease

Protein aggregation in the brain has been Protein aggregation in the brain has been observed in the brains of a big sort of diseases observed in the brains of a big sort of diseases characterized of progressive neurodegenetation, characterized of progressive neurodegenetation, such as such as

Alzheimer Disease (AD)Alzheimer Disease (AD)Parkinson Disease (PD)Parkinson Disease (PD)Huntington Disease (HD)Huntington Disease (HD)Creuzfeldt Jakob Disease (CJD) Creuzfeldt Jakob Disease (CJD) etc.etc.

Abnormal protein aggregations are associated Abnormal protein aggregations are associated with gene mutation, protein posttranslational with gene mutation, protein posttranslational aberrant modification, chronical viral infection in aberrant modification, chronical viral infection in the brain, brain aging and environmental toxin the brain, brain aging and environmental toxin as described as follows.as described as follows.

ALZHEIMER DISEASE (AD)ALZHEIMER DISEASE (AD) AD is a progressive, degenerative disorders of AD is a progressive, degenerative disorders of

uncertain causes, although abnormal uncertain causes, although abnormal metabolism and deposition of metabolism and deposition of ββ-amyloid protein -amyloid protein appears to be closely linked to pathogenesis.appears to be closely linked to pathogenesis.

AD is the most common cause of dementia. Its AD is the most common cause of dementia. Its incidence rises from less than 1% per year to incidence rises from less than 1% per year to more than 7% per year, and its prevalence more than 7% per year, and its prevalence from 3% to almost 50%, between the ages of from 3% to almost 50%, between the ages of 65 and 85 years65 and 85 years[[1313]]..

This translates into a prevalence of 10-20mil. This translates into a prevalence of 10-20mil. cases worldwide. Men and women are affected cases worldwide. Men and women are affected with equal frequency, when adjusted for agewith equal frequency, when adjusted for age[[13,1413,14]]..

AD is defined by characteristic histopathologic AD is defined by characteristic histopathologic features, especially neurofibrillary tangles and features, especially neurofibrillary tangles and neuritic (senile) plaquesneuritic (senile) plaques[[13,1413,14]]..

Examination of the brain will reveal variable Examination of the brain will reveal variable cortical atrophy with widening of the cerebral cortical atrophy with widening of the cerebral sulci that is most pronounced in the frontal, sulci that is most pronounced in the frontal, temporal and parietal lobe.temporal and parietal lobe.

The progressive loss of neurons in the entire The progressive loss of neurons in the entire cortex leads to slowly progressive mental cortex leads to slowly progressive mental deterioration. deterioration.

Clinical symptoms include the loss of recent Clinical symptoms include the loss of recent memory (the most common early sign) and loss memory (the most common early sign) and loss of long-term memory. The disease is progressive of long-term memory. The disease is progressive and leads to immobility in about ten yearsand leads to immobility in about ten years [[1414]]..

[[99]]

Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School.School.

PARKINSON DISEASE (PD)PARKINSON DISEASE (PD) Parkinsonism occurs in all ethic groups, in the Parkinsonism occurs in all ethic groups, in the

United States and Western Europe it has a United States and Western Europe it has a prevalence of 1-2/1000 population, with an prevalence of 1-2/1000 population, with an approximately equal sex distribution. The approximately equal sex distribution. The disorder becomes increasingly common with disorder becomes increasingly common with advancing age. advancing age.

It is characterized by expressionless face It is characterized by expressionless face (masklike), tremor 4-6Hz („pill-rolling“), (masklike), tremor 4-6Hz („pill-rolling“), hypokinesia, rigidity, and abnormal gait and hypokinesia, rigidity, and abnormal gait and postureposture[[13,1413,14]]..

It is important to compare HD with excessive It is important to compare HD with excessive movement to PD with decreased movementmovement to PD with decreased movement [[1313]]..

Etiology (multiple)Etiology (multiple)Idiopathic (most common)Idiopathic (most common)PostencephalitisPostencephalitisAutoimmune dysfunctionAutoimmune dysfunctionTraumaTraumaDrugs (illicit drug MPTP).Drugs (illicit drug MPTP).

LocationLocationSubstantia nigra and other brainstem Substantia nigra and other brainstem centers, cell loss in the globus pallidus and centers, cell loss in the globus pallidus and putamen and the presence of Lewy Body, putamen and the presence of Lewy Body, containing containing αα-synuclein in the basal ganglia, -synuclein in the basal ganglia, brainstem, spinal cord, and sympathetic brainstem, spinal cord, and sympathetic ganglia.ganglia.

ProcesProcesDecreased dopamine in corpus striatum.Decreased dopamine in corpus striatum.

TreatmentTreatmentDopamine agonists.Dopamine agonists.

HUNTINGTON DISEASE HUNTINGTON DISEASE (HD)(HD)

HD is a hereditar disorders of the nervous HD is a hereditar disorders of the nervous system characterized by the gradual onset and system characterized by the gradual onset and subsequent of chorea and dementia. It occurs subsequent of chorea and dementia. It occurs throughout the world and in all ethnic group. Its throughout the world and in all ethnic group. Its prevalence is about 5 per 100 000 populationprevalence is about 5 per 100 000 population[[1313]]..

HD is an autosomal dominant disorder. HD is HD is an autosomal dominant disorder. HD is caused by the increase in the length of a CAG caused by the increase in the length of a CAG triplet repeat present in a gene located on triplet repeat present in a gene located on chromosome 4p16.3. The product of the chromosome 4p16.3. The product of the mutated gene is an abnormal protein - mutated gene is an abnormal protein - HUNTINGTINEHUNTINGTINE[[99]]. .

The typical onset of HD appears usually in the The typical onset of HD appears usually in the 4th decade (typically between 30 and 50 years 4th decade (typically between 30 and 50 years of age), juvenile and late-onset cases are rare. of age), juvenile and late-onset cases are rare.

First symptoms are motor or mental changes First symptoms are motor or mental changes (choreatic dyskinesias, later dystonic or rigid (choreatic dyskinesias, later dystonic or rigid symptoms, personality and behavioral changes, symptoms, personality and behavioral changes, affective disorders, progressive cognitive deficits affective disorders, progressive cognitive deficits and later dementia)and later dementia)[[9,139,13]]. .

The pathophysiology of HD relates to The pathophysiology of HD relates to degeneration of GABA neurons (typical inhibitory degeneration of GABA neurons (typical inhibitory neurons) in the striatum. Decreasing functioning neurons) in the striatum. Decreasing functioning of inhibitory neurons leads to increased of inhibitory neurons leads to increased movementsmovements[[1414]]. .

[[99]]

Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School.School.

Material and MethodsMaterial and Methods--The Present The Present StateState

STEP 1 Detection of ER Single Imunohistochemistry RT-PCR Immunoblot - Western Blot

STEP 2 Designe of Primer ERα, ERβ(mus musculus and rattus norvegicus)

STEP 3 Isolation of mRNA from classical and non-classical estrogen target tissues and RT PCR

STEP 4 Construction of vector pEGFP-C2- ERα, ERβ

STEP 5 Sequencing of pEGFP-C2- ERα, ERβ, amplification

STEP 6 Transfaction into cell line HEK 293

Present State

Single Single IImunohistochemistrymunohistochemistry

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CEREBRAL CORTEX AREA

*

*

S1Tr/S1BF magn.20 x

Expression of ER in the cerebral cortex area have been verify by RT PCR & Western Blot.

CEREBRAL CORTEX AREA - RT-PCR RT-PCR

DDetection of ERetection of ERαα

+

+

This project was supported by scholarship cooperation This project was supported by scholarship cooperation

Ministry of Education Czech Republic and Ministry of Ministry of Education Czech Republic and Ministry of

Education P. R. of ChinaEducation P. R. of China in the Tongji Medical College in the in the Tongji Medical College in the

year 2005/06.year 2005/06.

I would like to thank my I would like to thank my supervisor supervisor Prof.Prof. LI He LI He,, PhD. PhD.,, for h for hisis expert advice and expert advice and great great help, help, Shen Shen HuangShen Shen Huang for hfor herer devoted devoted guidance relentless energyguidance relentless energy, , enthusiasmenthusiasm,, with which with which theythey guided guided me through the entire duration of my studme through the entire duration of my study in the Tongji Medical y in the Tongji Medical College.College.

Last but by no means the least; I must thank Last but by no means the least; I must thank my my collegues from collegues from Division of Histology, Tongji Medical College Division of Histology, Tongji Medical College for their for their enormous tolerance and support.enormous tolerance and support.

AcknowledgementsAcknowledgements

ReferencesReferences1.1. Gustafsson, A.J. (1999) Estrogen receptor Gustafsson, A.J. (1999) Estrogen receptor ββ– a new dimension in estrogen mechanism of action. – a new dimension in estrogen mechanism of action.

Journal of Endocrinology 163, 379–383.Journal of Endocrinology 163, 379–383.2.2. Liqin, Z., Tzu-wei, W., Roberta, D.B. (2004) Estrogen receptor subtypes alpha and beta contribute to Liqin, Z., Tzu-wei, W., Roberta, D.B. (2004) Estrogen receptor subtypes alpha and beta contribute to

neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain Research. neuroprotection and increased Bcl-2 expression in primary hippocampal neurons. Brain Research. Feb. 1010:22–34.Feb. 1010:22–34.

3.3. Pinzone,J.J et al. (2004) Molecular and Cellular Determinants of Estrogen Receptor ß Expression. Pinzone,J.J et al. (2004) Molecular and Cellular Determinants of Estrogen Receptor ß Expression. Molecular and cellular biology, June, 24(11): 4605–4612. Molecular and cellular biology, June, 24(11): 4605–4612.

4.4. Greenstein, B., Greeinstein, A. Color Atlas of Neuroscience: Neuroanatomy and Neurophysiology. Greenstein, B., Greeinstein, A. Color Atlas of Neuroscience: Neuroanatomy and Neurophysiology. Sttutgart: Thieme SRN, 2000. ISBN 0-086577-710-1.Sttutgart: Thieme SRN, 2000. ISBN 0-086577-710-1.

5.5. Hart, S.A., Patton, J.D., Woolley, C.S. (2001) Quantitative analysis of ER alpha and GAD Hart, S.A., Patton, J.D., Woolley, C.S. (2001) Quantitative analysis of ER alpha and GAD colocalization in the hippocampus of the adult female rat. J Comp Neurol. Nov 12;440(2):144-55.colocalization in the hippocampus of the adult female rat. J Comp Neurol. Nov 12;440(2):144-55.

6.6. Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz , S., Lagercrantz, J., Fried, G., Nordenskjold, Enmark, E., Pelto-Huikko, M., Grandien, K., Lagercrantz , S., Lagercrantz, J., Fried, G., Nordenskjold, M., Gustafsson, J.A. (1997) Human estrogen receptor ß – gene structure, chromosomal localization, M., Gustafsson, J.A. (1997) Human estrogen receptor ß – gene structure, chromosomal localization, expression pattern. Journal of Clinical Endocrinology and Metabolism 82:4258–4265.expression pattern. Journal of Clinical Endocrinology and Metabolism 82:4258–4265.

7.7. Rissman, E.F., Wersinger, S.R., Fugger, H.N., Foster, T.C. (1999) Sex with knockout models: Rissman, E.F., Wersinger, S.R., Fugger, H.N., Foster, T.C. (1999) Sex with knockout models: behavioral studies of estrogen receptor alpha. Brain Res. Jul 17;835(1):80-90. behavioral studies of estrogen receptor alpha. Brain Res. Jul 17;835(1):80-90.

8.8. Lundholm et al. (2004) Gene expression profiling identifies liver X receptor alpha as an estrogen-Lundholm et al. (2004) Gene expression profiling identifies liver X receptor alpha as an estrogen-regulated gene in mouse adipose tissue.regulated gene in mouse adipose tissue. Journal of Molecular Endocrinology.Journal of Molecular Endocrinology. 32 (3):879-892. 32 (3):879-892.

9.9. Roth,J., Zidovska,J., Uhrova,T., Doubek,P. at al Huntington´s disease and ethical problems related to Roth,J., Zidovska,J., Uhrova,T., Doubek,P. at al Huntington´s disease and ethical problems related to the diagnosis. Europ: Psychiatry 2000;suppl. 2:395–396.the diagnosis. Europ: Psychiatry 2000;suppl. 2:395–396.

10.10. Hoon,R. (2006) Hoon,R. (2006) Estrogen Regulation of Mitochondrial Transcription in HD. Boston University Medical Estrogen Regulation of Mitochondrial Transcription in HD. Boston University Medical Campus, dept. Of neurology and National Institute of Neurological Disorders and Stroke at the link: Campus, dept. Of neurology and National Institute of Neurological Disorders and Stroke at the link: http://researchresources.bumc.bu.edu/abstract/1R01NS052724-01.htm http://researchresources.bumc.bu.edu/abstract/1R01NS052724-01.htm

11.11. Paxinos, G., Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordination. The Prince of Wales Paxinos, G., Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordination. The Prince of Wales Medical Research Institute, Australia and McGill University Montreal, Quebec, Canada. San Diego: Medical Research Institute, Australia and McGill University Montreal, Quebec, Canada. San Diego: Academic Press, USA, 2001. ISBN 0-12-547636-1.Academic Press, USA, 2001. ISBN 0-12-547636-1.

12.12. Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School. At the link: Johnson,K.A., Becker, J.A. The Whole Brain Atlas. Harvard Medical School. At the link: www.med.harvard.edu/AANLIB/home.htmlwww.med.harvard.edu/AANLIB/home.html

13.13. Brown,E. Basic Concepts in Pathology. Beijing: McGraw-Hill 2002 ISBN 7-81071-389-2 Brown,E. Basic Concepts in Pathology. Beijing: McGraw-Hill 2002 ISBN 7-81071-389-2 14.14. Greenberg, D., Aminof, M., Simon, R. Clinical neurology. McGraw-Hill 5.th edition 2002 ISBN 7-117-Greenberg, D., Aminof, M., Simon, R. Clinical neurology. McGraw-Hill 5.th edition 2002 ISBN 7-117-

4950-2 4950-2

…IN A PICTURE

Cell with estrogen receptors, estrogen, and helper proteins.

A Estrogen receptorB EstrogenC Estrogen helper proteinsD nucleusE DNA genetic material

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ESTROGEN RECEPTOR

Formation of a recombinant DNA moleculeAn example of DNA cloning using bacterial plasmids

Protocol for cloning eukaryotic DNA fragments in lambda phage

Division of HistologyDivision of HistologyTongji Medical College, P.R. of Tongji Medical College, P.R. of

ChinaChina

LABORATORY OFLABORATORY OFTongji Medical CollegeTongji Medical College

Department of AnatomyDepartment of AnatomyLeipzig University, GermanyLeipzig University, Germany

Molikova Radka Bezdickova Marcela

Thank youThank you

for your attention!for your attention!